Population-based and translational studies provide evidence supporting the link between chronic hyperactivity, amyloid abnormalities, and memory impairments. Importantly, network hyper-excitability and reduced capacity of glutamate (Glu) transport is an early event and precedes the onset of Ab plaque/NFT pathologies and memory impairment (Masliah et al.,1996; Quiroz et al.,2010). Furthermore, hyper- synchronous network activity is particularly pronounced in families with presenilin (PS) mutations, with ~ 30% of such AD patients displaying epileptic seizures (convulsive and non-convulsive), and ~75% suffering from seizures in cases of the most aggressive PS variants (Larner and Doran, 2006; Kazim et al., 2017). During the previous grant cycles we have established a unique PS1 conformation-sensitive FRET-based assay in intact/live cells and demonstrated that fAD mutant PS1 adopts pathogenic conformation favoring generation of the longer Ab species (Berezovska et al.,2005, Uemura et al.,2009), and showed that similar changes occur in the wild type PS1 during aging and in sporadic AD (Wahlster et al., 2013). In our search for PS1- modulating interactors we performed a proteomics screen of mouse brain lysates and identified GLT-1, a major glutamate transporter in the CNS, as a novel PS1 binding partner. We have validated the PS1-GLT1 interaction on endogenous level in mouse brain and in primary astrocytes and neurons (Zoltowska et al., 2018). Aim 1 will build on this observation, and will determine the physiological stimuli that modulate this interaction in both astrocytes and neurons, verify if the interaction is limited to GLT1 glutamate transporter, will establish the exact PS1/GLT1 interaction sites, and will determine whether fAD PS1 and APP eg cleavage cascade mutations (collaboration with Project 1) producing various naturally secreted Ab42/40/ 38/37 levels and ratios affect the PS1-GLT1 binding. Next, we will examine if there is a functional crosstalk between the PS1 and GLT1. Aim 2 will determine whether manipulation of the PS1/g-secretase expression level and activity, allosteric modulation of the PS1 conformation (SGSMs, collaboration with Project 3) and/or change in the Ab42/40/38/37 levels and ratios affect GLT1 localization, multimerization and, ultimately, glutamate uptake. We will also detail the molecular mechanism(s) by which PS1 may modulate GLT1 cell surface trafficking and activity. Conversely, Aim 3 will examine whether genetic or pharmacologic manipulation of the GLT1 expression and binding to PS1 modifies PS1 conformation, APP processing/Ab generation, as well as the integrity of dendritic spines/synaptic markers. Understanding the precise physiological and pathological role of the novel PS1-GLT1 interaction is important because manipulation of this interaction may modify both perisynaptic glutamate uptake and Ab generation, and thus translate into ?dual-effect? therapeutics, targeting both glutamate overload and amyloid induced neurotoxicity.